Viral Diseases Flashcards
1
Q
HIV Size and Shape
A
Sphere-shaped enveloped retrovirus Gp120/gp41 protein complex anchored into the envelope
2
Q
HIV Genetics
A
Two identical copies of ssRNA, Codes 3 enzymes
- gag (p24, p40, p55): precursor for structural proteins
- pol (p51 reverse transcriptase, p66): Rt and replictaion enzymes (integrase and protease)
- env (gp41): glycoproteins in envelope for fuiosn and attachment
3
Q
Mechanism of HIV infection
A
- Targets of HIV: T cells, monocytes, macrophages, NKC, dendritic cells of lymph nodes and organs such as microglial cells of brain
- Adsorption of gp120 onto CD4 receptor; requires CXCR4 co-receptor for entry into T-cell, and CCR5 & CCR2 co-receptors for entry into macrophages
- After entry, viral RNA transrcibed into DNA which integrates with host DNA
- Active infection by viral replication and budding of virions
- Alternatively, HIV remains hidden as provirus in latent infection
- Evades immune system by cell-cell fusion, latent virions in vacuoles inside the cell etc.
4
Q
Course of HIV Infection
A
- Acute (Early): high biral replication, flu/mono-like symptoms about 3-6 wks after exposure
- Latent phase: low viral levels as it cleared from circulation, with no apparent symptoms, lasts up to 10 years, CD4 cells gradually destroyed
- Clinical AIDS stage: profound immunosuppression, high virla replication, absolute Th count of <200/µL
5
Q
Laboratory Results of HIV
A
Initial viral replication results in increased levels of p24 antigen and viral RNA in host’s blood Decreased CD4 T cell population
6
Q
HIV Laboratory Monitoring
A
Four types of tests used in diagnosis and monitoring of HIV infection:
- CD4 Tcell enumeration
- HIV antibody detection
- HIV antigen detection
- HIV nucleic acid detection
7
Q
CD4 T cell enumeration
A
- Reported as percentage, absolute cell count, or ratio of CD4:CD8 cells
- If cell count falls below 200/µL, patient is classified as having AIDS (normal range 500-1300/µL)
- Decrease in CD4 count results in an inverted ratio of less than 1:1 for CD4 to CD8 cells (normal is 2:1)
8
Q
HIV Antibody Detection
A
- Standard screening method is ELISA
- Standard confirmatory method is Western blot
- Other tests include agglutination tests, dot-blot test, home test kits etc.
9
Q
HIV Antigen Detection
A
p24 antigen testing by a solid-phase antigen capture enzyme immunoassay (EIA) P24 antigen becomes undetectable as host produces antibodies, hence can’t be used as screening test like ELISA
10
Q
HIV Nucleic Acid Detection
A
Viral load testing by RT-PCR, branced chain DNA assay (bDNA), and nucleic acid sequence based amplification (NASBA) Standard RT-PCR detects 400-750,000 copies of HIV RNA per ml of plasma.
11
Q
HIV Screening with ELISA
A
Detects HIV antibody in serum to viral antigens coated onto a solid support
Types of ELISAs:
- 1st generation: detects Abs to HIV-1 only
- 2nd generation: detects Abs to both HIV-1 and HIV-2
- 3rd generation: sandwich method, Abs in serum bind to recombinant HIV-1 and HIV-2 proteins coated onto wells, after washing enzyme-labelled HIV 1 and 2 antigens added which bind to already bound HIV-specific Abs
- 4th generation: simultaneously detects HIV-1 and HIV-2 Abs plus p24 antigen
12
Q
Problems with ELISA Screening in HIV
A
False-negative: Serum tested too early, immunosuppressive therapy, hypogammaglobulinemia, Improper handling of kits/reagents
False-positive: Repeated freeze-thawing of specimens, autoreactive antibodies, Multiple pregnancies, Severe hepatic disease, Malignancies, Passive immunoglobulin therapy
13
Q
Positive HIV Confirmations
A
When any serum sample comes ELISA positive for HIV, repeat the ELISA for a second time for presence of HIV-antibodies in patient’s serum.
Second-time positive cases are confirmed by Western blot/HIVD
Immunoassay for separated HIV antigens (p24, p31, and gp41 or gp120) treated with patient serum. Western blot is considered positive when at least two out of three possible antibodies are detected in patient’s serum.
14
Q
Vaccine Models
A
- Recombinant Protein Vaccine: HIV gp120 protein vaccine, marketed as AIDSVAX is in Phase III trials
- Safer to use Attenuated Viral Vaccine: Can trigger both humoral and cellular IR
- Risk of reversion DNA Vaccine: Plasmid based DNA vaccine IR produced not protective enough
- Prime Boost Strategy: Marketed as “DNA plus MVA” vaccine candidate contains genetically engineered plasmid plus modified vaccinia Ankarastrain virus
15
Q
Current HIV Treatment
A
- Highly Active Anti-retroviral Therapy (HAART)
- Reverse transcriptase inhibitors
- Protease inhibitors
- Integrase inhibitors
- Fusion inhibitors
16
Q
Liver Function Tests (Hepatitis)
A
- Bilirubin
- Alanine Aminotransferase (ALT)
- Aspartate Aminotransferase (AST)
Serum levels are elevated in proportion to the amount of liver damage (enzymes are contained within the hepatocytes, inflammation causes their release; Bilirubin rises as liver damage continues)
17
Q
Acute Viral Hepatitis Tests
A
- anti-HAV: an IgM antibody directed against Hepatitis A (HAV)
- HBsAg (hepatitis B surface antigen),
- anti-HBc: an IgM antibody directed against the hepatitis B core antigen
- anti-HCV: antibody to Hepatitis C (HCV)
18
Q
Hepatitis A
A
- Route of Transmission: Fecal-oral
- Chronic Infection: No (acute)
- Prevention: pre/post exposure immunization
- Member of the picornavirus family; various serotypes include rhinoviruses (common cold)
- Non-enveloped – strand RNA packaged within capsids with icosahedral symmetry
- Only hepatitis virus that can be propagated in tissue culture
19
Q
Hepatitis B
A
- Route of Transmission: Percutaneous, Permucosal
- Chronic Infection: Yes
- Prevention: pre/post exposure immunization
- Long incubation period
20
Q
Hepatitis C
A
- Route of Transmission: Percutaneous, Permucosal
- Chronic Infection: Yes
- Prevention: Blood Donor Screening, Behavior Modification
- Longer incubation than A but shorter than B
- Typically asymptomatic but high rate of progression to cirrhosis and HPC (Hepatic Cancer)
21
Q
Hepatitis D
A
- Route of Transmission: Percutaneous, Permucosal
- Chronic Infection: yes, low risk with coinfection
- Prevention: pre/post exposure immunization, behavior modification
- Coinfection needed with Hepatitis B
22
Q
Hepatitis E
A
- Route of Transmission: Fecal-oral
- Chronic Infection: No (acute)
- Prevention: Ensure safe drinking water
- Average incubation: Average 40 days, Range 15-60 days
23
Q
Causes of Hepatitis A Infection
A
From close personal contact, child care, nursing homes, contaminated food, water HAV infects cells of the intestinal mucosa and, following replication, gains access to blood, then is delivered to Liver cells to replicate, the viral progeny accumulate in bile fluid and are delivered back to the GI tract
24
Q
ALT
A
Alanine aminotransferase, is a hepatocyte-specific activity that is released into circulation following hepatocellular necrosis
Used as marker for disease progression
25
Hepatitis B Panel
This panel consists of four hepatitis B markers:
* HBsAg
* HBeAg
* anti-HBe
* anti-HBs
* IgM antibody to the core antigen (anti-HBc IgM) may also be included
26
Markers for Chronic Hepatitis Infections
* Three markers are used to determine the stage of chronic infection: HBsAg, HBeAg, and anti-HBc total
* HBsAg and anti-HBc total will almost always be present
* Depending on the stage of the disease, HBeAg may or may not be present,
* Chronically infected individuals with HBeAg typically have higher viral loads
27
HBV Vaccinations
* 1st generation HBV vaccine: surface antigen purified from chronically-infected individuals and inactivated by a variety of techniques. Still used in many third world countries
* Current vaccine: HBsAg subunit vaccine produced in yeast
28
Immunity Testing in HBV
* Anti-HBs (antibody to the surface antigen) is the only marker for determining immunity to a HBV
* Anti-HBs appears early and the titer may eventually decline.
29
Percutaneous Transmission of HCV
* Injecting drug use
* Clotting factors before viral inactivation
* Transfusion, transplant from infected donor
* Therapeutic (contaminated equipment, unsafe injection practices)
* Occupational (needlestick)
30
Perinatal Transmission of HCV
Transmission only from women HCV-RNA positive at delivery
No association with:
* Delivery method
* Breastfeeding
31
Mononucleosis Facts
* Widely disseminated, ubiquitous
* 95% of the world’s population is exposed to this virus
* Adults: usually have protective antibodies to EBV
32
EBV
* EBV is member of the herpes family of virus, double-stranded DNA virus
* EBV infects B-lymphs and epithelial cells, infection initiates in the throat, in an acute, benign,and self-limiting lymphoproliferative condition
33
Viral Replication of EBV
* Adsorption: the virus attaches to a cell- surface protein which functions as a viral receptor (usually a B lymphocyte)
* Penetration and uncoating: the virus moves into the cell where the viral envelope then fuses with the vesicle membrane and releases the viral contents into the cell’s cytoplasm.
* Establishment of latent infection
34
EBV Antibody Detection
1. VCA viral capsid Ag (IgM (early infection) IgG (lifelong antibody))
2. EA early antigen (EA IgG: acute & Burkitt’s lymphoma)
3. EBNA nuclear antigens (binds to the viral DNA and allows the EBV genome to be maintained in the B cell as a circular DNA episome) in convalescence
35
Infection Phases in EBV
* Primary infection: symptoms fever, malaise, sore throat, enlarged neck lymph nodes, splenomegaly.
* Incubation: 10-50 days, lasts for one to four weeks
* If lingers more than 6 months becomes chronic EBV infection, chronic fatigue syndrome, **Hepatitis** is a common complication
* X-linked lymphoproliferative syndrome: a rare inherited disorder: develops a fatal primary infection.
36
EBV Immune Response
* Infection with EBV results in both humoral and cellular immunity to the virus.
* Antibodies directed against viral structural proteins and EBNA is diagnostic for infection.
* Cellular immune response is important for the control of EBV infection
* Natural killer cells and CD4 & CD8 cytotoxic Tcells control the proliferation of EBV infected B cells during the primary infection
* After recovery from an acute infection, HLA restricted cytotoxic T ells are important in controlling EBV
37
EBV Effect on Cells
* Atypical lymphocytes in mono are primary T cells, many of which are responding the EBV infected B cells.
* Activation of the B-cells by EBV results in production of antibodies, causing elevated titers of heterophile antibodies, and sometimes cold agglutinins, cryoglobulins, rheumatoid factors, and antinuclear antibodies.
* Chronic Active EBV infection: 3 features: severe illness of more that 6 months, histological evidence of organ disease (lungs, liver, bone marrow) and demonstration of EBV antigens or DNA in tissue.
38
EBV Assoc. Diseases
1. Nasopharyngeal Carcinoma
2. Burkitt’s Lymphoma
3. Hodgkin’s disease
4. Lymphoproliferative disease
5. Neoplasms of the thymus, parotid gland, and supraglottic larynx
6. Can cause complications involving the cardiac, ocular, respiratory, hematologic, digestive, renal, and neurologic systems
7. Neurologic syndromes: bells palsy, Guillian Barre syndrome, meningoencephalitis, Reye's syndrome, myelitis, cranial nerve neuritis and psychotic disorders
39
Nasopharyngeal Carcinoma and EBV
100% of these cases expresses the EBV proteins, have elevated titers of IgA antibody to EBV.
40
Burkitt's Lymphoma and EBV
A malignant lymphoma of the small B cells, particularly in Africa. Infection with malaria is thought to diminish the Tcell control of proliferating EBV infected B cells.
41
Hodgkin's Disease and EBV
Patients often have high titers of antibody to EBV structural proteins before the onset of the lymphoma.
42
Lymphoproliferative Disease and EBV
(AIDs or congenital): these patients have impaired T cell immunity and are unable to control the proliferation of EBV infected B cells and have elevated levels of interleukin6, a B cell growth factor that may increase the proliferation of EBV B cells.
43
Clinical Diagnosis of EBV Using Antibodies
* Helpful in determining acute or past infection
* VCA: viral capsid antigen IgM & IgG primary infection presence of IgM accompanied by rising IgG-VCA levels. Produced by infected B lymphs
* EA: early antigen IgG not detected in about 20% of EBV infections, present in Burkitt’s lymphoma or nasopharyngeal carcinoma
44
Heterophile Antibodies and EBV
Heterophil Ab of IM: IgM type of antibody, usually appears during the acute phase, the ag that stimulates its production is unknown.
Has these characteristics:
1. Reacts SRBC, horse, ox red blood cells
2. Absorbed with beef red blood cells
3. Not absorbed with Forssman Ag (guinea pig kidney cells), Does not react with EBV-specific ag
45
Paul-Bunnell test for IM Ab
* Presumptive Testing, using Paul-Bunnell glycoproteins (antigens) on red cell membranes
* Pt serum + SRBC = agglutination
* If heterophile Ab present, it is not specific for IM Ab
46
Davidsohn Differential test for IM Ab
Differential Testing highly specific for IM
Pt serum + guinea pig kidney/beef rbc=
1. Antigen from guinea pig kidney cells which are rich in Forsmann ag will absorb Forssman AB and serum sickness Ab
2. Beef erythrocytes are poor in Forsmann ag, but absorb the heteorphil ab of IM.
3. Will only partially remove IM Abs.
4. Absorption is detected by loss of ability to agglutinate SRBC
47
Definition of a heterophile antibody
Stimulated by one antigen and reacts with an entirely unrelated surface antigen present on cells
48
Summary of mononucleosis test by latex agglutination
Tests for the IgM heterophile antibody by using agglutination of sheep red blood cells, Forssman antibodies may be absorbed out with a guinea pig kidney antigen
Positive tests are associated with hepatitis, rubella, leukemia, rheumatoid arthritis, Burkitt's lymphoma and other pathological conditions
49
Mono spot test interpretation
1+ small clumping with opaque fluid in background
2+ moderate clumping with fluid slightly opaque and background
3+ large clumping with clear background
50
Early EBV infection
When primary infection is delayed until adulthood, there's a 50% chance that it will occur with classic clinical manifestations of mononucleosis
51
Early EBV infection antigens
EBV induced nuclear antigen (EBNA), EBV induced early antigen (EA), viral capsid antigen (VCA), and EBV induced membrane antigen (MA) IgM antibodies to VCA are detectable in the early course of disease
52
Rubella
* Single-stranded RNA virus, two viral envelopes
* Causes German or 3-day measles
* Symptoms: rash, fever, malaise, upper respiratory infectious route
* Age: 5-14yr olds
* Congenital rubella: maternal infection can lead to congenital defects - CHF, hepatitis, low birth weight, diabetes
53
Rubella Laboratory Diagnosis and Vaccination
* Latex agglutination and EIA
* Titer of 1:8 is considered immune, a 4-fold increase in titer with symptoms indicates recent rubella infection
* Vaccination: in 1962-1965, worldwide epidemic lead to immune program-new strains developed
54
Cytomegalovirus (CMV)
* Large, enveloped DNA virus, ubiquitous
* Spreads from cell to cell
* Transmitted through prolonged close contact with infectious body fluids (ie. Sexual contact to transfusion/transplantation)
* Acquired CMV is usually asymptomatic, occasionally it is self- limited, heterophile negative, mono-like syndrome with sore throat, fever, malaise. In latent disease, virus can reactivated with appropriate stimuli
55
Patients at risk for CMV Infection
Immunocompromised, HIV pos, or transplant patients
56
HSV1 and HSV2
* Herpes Simplex Virus 1: causes cold sores, mainly in the mucosal membranes of the mouth
* Herpes Simplex Virus 2: watery blisters on mucous membranes of genitals
* Both become latent and have periodic reactivation
57
Laboratory Testing of HSV1 and HSV2
* Viral culture from swabs of lesions, using direct immunofluorescence of the Herpes Ag.
* Serological tests require a significant titer rise to be useful - PCR for HSV nucleic acid
58
Chicken Pox/Shingles Lab Testing
* Direct immunofluorescence of the Herpes Ag from lesion scrapings
* PCR for VZV DNA
* FAMA (Fluorescent Ab to Membrane Ag)
59
Herpes Virus Facts
* DNA virus, HSV- 2 identified as genital virus
* Transmitted perinatally if mom is in active state at time of delivery, can be fatal to infant
60
Appearance of Serological Markers in Hep. B
61
Congenital Rubella
Can cause deafness, eye defects, cardiac abnormalities, mental retardation, and motor disabilities
62
Laboratory Testing of Rubella
* Hemagglutination inhibition
* Passive hemagglutination
* Complement fixation
* Latex agglutination
* Immunoassays
63
HBsAg
First to appear (2-10 wks), peaks in acute phases then declines as Pt developns Ab
Indicates active infection
64
HBeAg
Appears after HBsAg and disappears slightly before it in recovering patients, appears during periods of high replication
65
IgM HBc
Current/recent infection (1-2 wks post HBsAg), persists in high titers for 4-6 mos.
66
IgG HBc
Appears before IgM HBc is gone and persists for life
67
anti-HBe
Shortly after HBe disappears, indicates recovery
68
CMV Symptoms
Fever, myalgia, and fatigue
69
CMV Laboratory Testing
Viral Culture, Viral Ag assays, Molecular assays and Serology
70
Rubeola Symptoms
Causes Measles
* Characteristic grey/white lesions in mouth surrounded by highly inflamed tissue
* Fever
* Cough
* Runny nose
* Conjunctivitis
71
Ig Testing in Rubeola
IgM is more frequently looked for due to its indication of early infection and low risk of false positives with ELISA
72
Laboratory Testing of Rubeola
* ELISA
* Hemagglutination inhibition
* Microneutralization
* Complement fixation
* Indirect fluorescent Ab tests
* PCR
73
Symptoms of Mumps
Inflammation of Parotid Glands
74
Rubeola Virus
* Single-stranded RNA virus
* Enveloped
* Genus Morbillivirus
75
Mumps Virus
* Enveloped
* Single-stranded RNA virus
* Rubulavirus genus
76
DNA Viruses
* EBV
* CMV
* Herpes
77
RNA Non-enveloped Viruses
Hepatitis A and E
78
RNA Enveloped Viruses
* HIV
* Rubella
* Rubeola
* Mumps
* Hepatitis B, C, D
